1. Field of the Invention
With the increase in emphasis within the agricultural community on using biocontrol agents as substitutes for chemicals in the control of weeds, insects, and other pests, considerable research has been directed to compositions and mechanisms for delivering the agents in a way which will preserve their effectiveness in the field. This invention relates to a novel granular formulation of biocontrol agents.
2. Description of the Prior Art
Dunkle et al. [Environ. Entomol. 17:20-126 (1988)] and U.S. Pat. No. 4,859,377 show a granular formulation of Bacillus thuringiensis (B.t.) encapsulated within a starch matrix. The advantage of this product over prior formulations was that it allowed incorporation of various additives such as sunlight protectors and palatable feeding stimulants, thereby reducing the amount of active ingredient necessary for control. Trimnell et al. [J. Controlled Release 7: 263-268 (1988)] reported a sprayable herbicide formulation utilizing pregelatinized corn starch and flour. These sprays were designed to coat plant leaves with a thin film of material which would autoencapsulate (encapsulate the active agent in situ) upon drying and thereby allow sustained release of active ingredient. However, within 2-3 days after application, these films were found to peel away from the plant leaves. In general, sprayable formulations of B.t. lose activity within 2-4 days following application to plant foliage in the field [Morris, Can. Ent. 115: 1215-1227 (1983); Beegle et al., Environ. Entomol. 10: 400-401 (1981); Leong et al., Environ. Entomol. 9: 593-599 (1980)].
Shasha et al. [U.S. Pat. No. 5,061,697 and J. Econ. Entomol. 83(5): 1813-1817 (1990)], teach sprayable, starch-based formulations for autoencapsulating biological control agents, such as pathogenic bacteria and viruses. These compositions incorporate a sugary material to promote adherence of the encapsulated agent to treated foliage.
Walker et al., U.S. Pat. Nos. 4,718,935 and 4,767,441, teach pelletization of infective propagules of fungal plant pathogens using aqueous solutions of sodium alginate and calcium chloride. This system relies upon the ability of the fungus to grow through the gelled alginate matrix to the pellet surface, where spores of the fungus are produced and released to the target plant.
Quimby et al. (U.S. Pat. No. 5,358,863) teach forming granules by encapsulating bacteria, fungi or nematodes useful for controlling agricultural pests in alginate, starch or wheat gluten, and then coating the granules with an invert oil that forms a water-in-oil emulsion and an absorbent for the oil to make the coated granules free-flowing. See also Amsellem et al. [Phytopathology, 80(10):925-929 (1990)]. The oil slows the drying of the organisms to maintain their vitality. The resulting products are applied by spraying through large-orifice nozzles.
McCabe et al. (U.S. Pat. No. 4,530,834) teach the preparation of entomopathogenic fungal insect control agents by culturing mycelia in a suitable medium, harvesting the growing mycelia, treating the mycelia with a protective agent such as maltose or glucose, and drying the product.
Mungier et al. [Appl. and Environ. Micro., 50(1): 108-114 (1985)] presents a study on the survival of bacteria and fungi in relation to water activity and the solvent properties of water in biopolymer gels. This reference shows that cells survive at a water activity (aw 0.069 and below and die at an aw above that.
Caesar et al. [Appl. and Environ. Micro., 57(1): 168-172 (1991)] demonstrated that strains of Pseudomonas and members of the family Enterobacteriaceae could be conditioned for improved shelf life in simple dry formulations by aging, exposure to osmotica, or growth on media amended with sucrose or betaine. Similarly, Leslie et al. [Appl. and Environ. Micro., 61(10): 3592-3597 (1995)] teach that when E. coli and B.t. were dried in the presence of trehalose or sucrose, their survival rate was greatly enhanced over organisms dried without the sugars. The increased survivability is attributed to lowering of the transition temperature (Tm) of the dry membranes by replacement of the water between the membrane lipid headgroups with the disaccharide.
Connick et al. [U.S. Pat. No. 5,074,902 and Biological Control 1: 281-287 (1991)] teach encapsulation of fungal propagules in a wheat gluten matrix, resulting in a pasta-like product referred to as xe2x80x9cPestaxe2x80x9d. Pesta is prepared from a dough of wheat flour, kaolin filler, fungus and water. The dough is rolled into a thin sheet, air-dried, and cut with a pasta maker or ground into granules. Upon application of the product to the soil, the fungi grow and sporulate on the granules. The Pesta technology has also been shown by Connick et al. [Journal of Nematology 25(2):198-203 (1993)] to be useful for the encapsulation of nematodes (Steinernema carpocapsae). In Biocontrol Science and Technology 6:277-284 (1996), Connick et al. show that, at relatively high aw, sucrose helps maintain Colletotrichum sp. conidia viability in Pesta granules during storage.
We have now unexpectedly discovered a simple and inexpensive procedure for preparing a stabilized, granular, biocontrol agent which can optionally be formulated as a rehydratable sprayable composition. The invention is applicable to a wide variety of living, pathogenic biocontrol agents useful in the management of all types of agricultural pests.
In accordance with this discovery, it is an object of the invention to provide a facile, universal, and industrially acceptable procedure for granular formulation of sensitive biocontrol agents.
It is also an object of the invention to formulate biocontrol agents without loss of viability and with a high degree of stability under storage and field conditions.
Another object of the invention is to prepare biocontrol products that are clean, easy to handle, and have relatively low crop phytotoxicity.
A further object of the invention is to package biocontrol agents into formulations that can be applied with conventional agricultural sprayers.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
The primary components of the formulations of the invention include (1) a biocontrol agent, (2) a water absorbent material, (3) a membrane stabilization agent, and (4) a granulating agent. Oil is an optional primary component. The Biocontrol Agent
The biocontrol agents contemplated for use herein include without limitation all bacteria, fungi, yeasts, viruses, microsporidians, protozoa, nematodes and other such organisms that are pathogenic toward target pests. Of course, any component of the organism or stage of its life cycle which is infective to the host upon contact or ingestion is considered to be within the scope of the invention. For instance, in the case of B.t., the vegetative cells, spores, and proteinaceous crystals are all effective in directly or indirectly killing host insects susceptible to B.t. It is also known that naturally occurring and synthetic vectors such as plasmids, phages, and various DNA/RNA constructs have potential for functionally modifying higher organisms, and therefore are also included herein as being within the scope of the term xe2x80x9cbiocontrol agent.xe2x80x9d Examples of other agronomically important pest pathogens besides B.t., without limitation thereto include: other entomopathogenic bacteria such as B. sphaericus, and B. popillae; plant pathogenic bacteria, such as Pseudomonas spp. and Agrobacterium; plant pathogenic fungi, such as Sclerotinia, Rhizoctonia, Fusarium, Alternaria, Colletotrichum, and Sclerotium; entomopathogenic fungi, such as Pandora, Beauveria and Conidiobolus and the yeasts; entomopathogenic viruses, such as Autographa californica nuclear polyhedrosis virus, and Heliothis spp. virus; microsporidians such as Vairimorpha necatrix and Nosema locustae, as well as the nematode Steinernema carpocapsae and the gall-forming nematode Subanguina picridis. 
The biocontrol agents of the invention are normally propagated by cultivation in a suitable aqueous medium and then recovered as a concentrated suspension of the biocontrol agent. Typically, these suspensions will comprise about 40-95% water. The Water Absorbent Material
Suitable water absorbent materials are those which are capable of absorbing several times their own weight in water, preferably, at least about 100 times their own weight in substantially pure water. Most notable are the starch polyacrlonitrile graft copolymers (e.g. the composition of U.S. Pat. No. 3,935,099, herein incorporated by reference) and similar starch graft copolymers which are commercially known by names such as xe2x80x9cSuper Slurperxe2x80x9d, Water-lock(copyright), etc. Upon absorbing water or other aqueous liquids, these materials swell into amorphous gels which tenaciously retain the absorbed water.
The amount of water absorbent material should be sufficient to absorb, and thereby bind, the free (available) water in the suspension of biocontrol agent. Typically, the ratio of water absorbent material to available water in the suspension would be on the order of about 1:5 to about 1:100, preferably about 1:10. In terms of the total weight of the final product, the water absorbent should constitute about 5-16% by weight. The skilled artisan will appreciate that salts in the suspension of the organism as well as other components already present in the formulation mixture at the time of addition of the absorbent material may reduce the inherent absorbancy of the material, requiring amounts in excess of that required for absorbing an equivalent amount of purified water. The relatively high absorbencies of these materials allows for binding of the free water using a relatively minor amount of this component, thereby permitting the formulation to accommodate effective amounts of the other components.
The Membrane Stabilization Agent
The principal function of the membrane stabilization agent is to bring the biocontrol agent to an immediate state of physiological quiescence and allow the organism to survive for long periods of time, even under adverse conditions. The preferred stabilization agent is sucrose; though it is envisioned that other disaccharides, such as trehalose, which have a similar capacity for reducing water availability could also be used. It is important that the membrane stabilization agent also have the property of forming a nongummy dough when mixed with the biocontrol agent and the water absorbent material described above.
The amount of the membrane stabilization agent should be in the range of about 50 mM to about 1 M, or about 10-65% by dry weight of the complete formulation. At 60% sucrose, the water potential of the formulation drops to at least xe2x88x9275 bars.
The Granulating Agent.
After the primary components heretofore described are blended into a dough, it is necessary to mix the dough with a granulation agent which causes the dough to break into small granules or xe2x80x9ccrumbsxe2x80x9d and renders the product flowable. The size of the granules can be tailored for a predetermined end use application by the nature and amount of the particular agent selected. For instance, siliceous materials such as diatomaceous earth, Cab-O-Sil(copyright) and Hi-Sil(copyright) tend to produce a small mesh granular product which, upon drying, can be readily resuspended into a sprayable formulation. Coarser agents such as corn cob grits, pregranulated starch, etc. will yield a larger mesh particulate product for field application in granular form.
The amount of the granulating agent will typically be in the range of about 5-20% by weight of the total product, with a preferred range of about 10-16% by weight.
Optional Oil Component:
Incorporating an oil into the formulation has been found to enhance the storage stability and field viability of certain living biocontrol agents. Moreover, the oil unexpectedly enhances the effectiveness of the granulation agent to convert the dough-like mass into discrete particles. Suitable oils include mineral oil and vegetable oils, such as those derived from corn, soybean, sunflower, safflower, rapeseed, cottonseed and the like. Usually the oil would constitute less than about 20% by weight of the total composition.
Formulation Protocol:
The biocontrol agent suspension, the water absorbent agent, and the membrane stabilization agent can be blended together in any order and by any conventional means in the art. In addition, the membrane stabilization agent can be prepared in an aqueous solution and used as a suspending agent for the biocontrol agent. The optional oil component is preferably added last, for the reason that it could otherwise interfere with the ability of the water absorbent to absorb the free water in the suspension as previously discussed.
The primary components are blended together into a dough-like mass in a Sigma mixer or the like, and thereafter the granulating agent is added. Blending is continued until the dough xe2x80x9ccrumbsxe2x80x9d and is recoverable as wet granules. The granules can thereafter be dried by any known method which will not adversely affect the viability of the biocontrol agent. Though air drying is usually preferred, under appropriate situations, mild oven drying could also be used. Thus, by appropriate selection of component ratios as within the skill of a person in the art, it is possible to recover particles of granulated biocontrol agent of the desired size without the need for grinding.
Other Components:
Besides the primary components described above, other additives and adjuncts may be formulated into the subject compositions. Examples of these include dispersants, feeding stimulants (phagostimulants), UV protectants, preservatives, and inert fillers. Also of interest are agronomically acceptable carriers or vehicles for the active agent or any of the other components formulated into the granular compositions.
In accordance with one embodiment of the invention, the granules are resuspended in water, an oil-in-water emulsion, or an invert emulsion (water-in-oil) for field application as a sprayable liquid. In yet another embodiment of the invention, the granulated biocontrol agent can be applied to the plant foliage or soil as a dry formulation. In either form, the flowable nature of the product lends itself to field application using conventional equipment without the need for specialized modification.
Pests:
The target pests contemplated for control by means of the subject granulated agents of the invention include all species susceptible to the above-mentioned biocontrol agents. Such pests include insects, weeds, crop diseases, detrimental nematodes and the like. The granulated agents find application for field crops, orchard crops, ornamentals and certain stored agricultural products.
The granulated agents of the invention may be stored at room temperatures for extended periods of time with minimal loss of vitality. For instance, the survival rate of Pseudomonas syringae granulated as described in the examples and stored at 22xc2x0 C. for 202 days was approximately 85%. At xe2x88x9215xc2x0 C., the survival exceeded 95%.
It should be understood that living organisms are very fragile and sensitive to environmental conditions. Even with the advantages achieved by the present invention, the skilled artisan will appreciate that some organisms will not be effective against specific targets and that the effectiveness of some granulated organisms will be a function of the specific environmental conditions. However, it would be within the skill of a person in the art to determine the effectiveness of candidate organisms and formulations for a particular end use application.